CN105754941B

CN105754941B - In-vitro induced amplification culture method for peripheral blood NK cells

Info

Publication number: CN105754941B
Application number: CN201610242598.4A
Authority: CN
Inventors: 陈继冰; 牛立志; 徐克成
Original assignee: Guangzhou Fuda Medical Co ltd
Current assignee: Guangzhou Fuda Medical Co ltd
Priority date: 2016-04-18
Filing date: 2016-04-18
Publication date: 2020-06-02
Anticipated expiration: 2036-04-18
Also published as: CN105754941A

Abstract

The invention provides a method for inducing, amplifying and culturing peripheral blood NK cells. The induced amplification culture method of the peripheral blood NK cells is characterized in that the peripheral blood mononuclear cells are cultured in a combined manner by applying cell factors interleukin-2, interleukin-15, 4-1BBL and SCGM culture media, interleukin-15 and 4-1BBL in the early stage are incubated and fixed on a cell culture plate to induce and activate a small amount of NK cells in the mononuclear cells to rapidly proliferate, interleukin-15 and 4-1BBL with solubility in the later stage induce a large amount of NK cells to rapidly proliferate, and then the cultured NK cells are collected to carry out cell purity and detection on the killing effect of tumor cells so as to ensure the anti-tumor effect of the NK cells. The method has simple and easy induced culture process, short period and low cost; the yield is high, and the repeatability is good; the cultured cells have good activity, and the number and the activity of the obtained NK cells can ensure the application of clinical tumor immunotherapy.

Description

In-vitro induced amplification culture method for peripheral blood NK cells

Technical Field

The invention belongs to the field of immunology and epigenetics research, and relates to an in vitro induction amplification culture method of peripheral blood NK cells.

Background

NK cells (natural killer cells) belong to immune cells of the innate immune system, and account for 10% -15% of peripheral blood lymphocytes, thereby constituting the first line of defense of the immune system of the organism. Since NK cells recognize target cells without MHC restriction, tumor cells can be directly killed in a state without sensitization beforehand. The NK cells can generate a series of cytokines while exerting immune effect, and adjust adaptive immunity, thereby becoming a bridge connecting the innate immunity and the adaptive immunity of the organism. Because NK cells have the characteristics of direct killing and immunoregulation, the NK cells are expected to become effector cells of adoptive immunotherapy of tumors. In addition, there are increasing reports of genetic engineering of primary NK or NK cell lines to improve tumor killing specificity and activity for adoptive immunotherapy. Therefore, the NK cells have wide application prospects in tumor biotherapy, and the problem that the clinical application of the NK cells is urgently needed to be solved is to enable more patients to receive NK cell immunotherapy, reduce the induced amplification culture cost of the NK cells, simplify the culture steps of the NK cells and improve the induction efficiency of the NK cells.

Disclosure of Invention

The invention aims to provide an in-vitro induced amplification culture method for peripheral blood NK cells, which can effectively reduce the culture cost of the NK cells and obtain a large amount of NK cells, and provides a basis for the application of the NK cells in the aspect of clinical tumor immunotherapy.

The in vitro induction amplification culture method of the peripheral blood NK cells comprises the following steps:

A. preparing human peripheral blood mononuclear cells by a conventional technology;

B. resuspending the collected mononuclear cells with a red blood cell lysate, and lysing to remove residual red blood cells;

C. resuspending the mononuclear cells by using 38ml of SCGM culture medium, adding 2ml of inactivated autologous serum and 2 ten thousand units of recombinant human IL-2, uniformly mixing, adding into a culture plate, and placing into an incubator at 37 ℃ for standing culture; the interleukin-15 and 4-1BBL required by the NK cells are cultured and activated in the culture plate in a fixed mode;

D. on day 3, 20ml of SCGM culture medium containing 1-2% of autologous serum, 1 ten thousand units of IL-2, a proper amount of interleukin-15 and a proper amount of 4-1BBL are supplemented, and the culture is continued after the mixture is uniformly mixed;

E. on day 4, the culture medium in the culture plate was transferred to a culture bag and further cultured, and 40ml of SCGM medium containing 1-2% of autologous serum and 2 ten thousand units of IL-2, an appropriate amount of interleukin-15 and an appropriate amount of 4-1BBL were supplemented to the culture bag every 3-4 days, and the total number of NK cells that could be obtained was up to 5.5X 10 for a total of 18 days⁹And a viable cell rate of about 90%, and wherein the purity of NK cells is about 80%.

According to a further feature of the method for culturing peripheral blood NK cells by in vitro induction and amplification of the present invention, in step B, mononuclear cells collected from each 40ml of peripheral blood specimen are resuspended in 5ml of erythrocyte lysate, and the remaining erythrocytes are completely lysed in a 37 ℃ water bath for 1 minute.

According to a further feature of the method for culturing peripheral blood NK cells by in vitro induced expansion of the present invention, in steps D and E, the interleukin-15 is supplemented at a concentration of 10 ng/ml.

According to a further feature of the method for culturing peripheral blood NK cells by in vitro induced expansion of the present invention, in the steps D and E, 4-1BBL is supplemented at a ratio of 10 ng/ml.

According to a further feature of the method for culturing peripheral blood NK cells by in vitro induced expansion, in step C, PBS containing interleukin-15 and 4-1BBL is added to the culture plate one day ahead of time and incubated at 4 ℃ for storage, and the unfixed interleukin-15 and 4-1BBL are washed away when the method is used.

Preferably, PBS containing 2. mu.g/ml interleukin-15 and 2. mu.g/ml 4-1BBL is added to the culture plate one day ahead for incubation and preservation at 4 ℃.

The induced amplification culture method of the NK cells has the following characteristics and advantages:

(1) the peripheral blood NK cell in-vitro induction amplification culture method provided by the invention is characterized in that peripheral blood mononuclear cells are cultured in a combined manner by applying cell factors interleukin-2, interleukin-15, 4-1BBL and SCGM culture media, interleukin-15 and 4-1BBL in the early stage are incubated and fixed on a cell culture plate to induce and activate a small amount of NK cells in the mononuclear cells to rapidly proliferate, interleukin-15 and 4-1BBL with solubility in the later stage induce a large amount of NK cells to rapidly proliferate, and then the cultured NK cells are collected to carry out cell purity and detection on the killing effect of tumor cells so as to ensure the anti-tumor effect of the NK cells. The purity of the NK cells is detected by detecting the growth state of the NK cells, and the cell number and cytotoxicity of the NK cells obtained by the method can be ensured to be used for clinical treatment.

(2) The invention determines that the optimal culture concentration of PBMC is induced and cultured by 10ng/ml of interleukin-15 and 10ng/ml of 4-1BBL, and the optimal culture concentration is determined by comparing different concentration combination schemes.

(3) The present invention determined that the optimal culture time was 14 days, which was determined by comparing the NK cell tumor killing rates at different culture times.

(4) The induced amplification culture method of the NK cells is simple and easy in process, short in period of induced proliferation of the NK cells and low in cost.

(5) By adopting the induced amplification culture method of the NK cells, the induction efficiency of the NK cells is high, and the repeatability is good.

(6) By adopting the induced amplification culture method of the NK cells, the activity of the proliferated NK cells is good, the number and the cytotoxicity of the NK cells can ensure the clinical immunotherapy of tumors, a foundation can be provided for the clinical application of the NK cells, and the method has good popularization value.

Drawings

FIG. 1 shows the total number of cells obtained after PBMC cells were induced for 14 days in different protocols.

FIG. 2 is a graph showing the state of cell growth of human peripheral blood mononuclear cells at day 0.

FIG. 3 is a diagram showing the state of cell growth 18 days after induction culture of human peripheral blood mononuclear cells.

FIG. 4 is a graph showing the total number of cells in human peripheral blood mononuclear cells after induction culture.

FIG. 5 shows the survival rate of human peripheral blood mononuclear cells after induction culture over time.

FIG. 6 is a diagram showing the flow analysis of NK cells (CD3-CD16+ CD56+) in human peripheral blood mononuclear cells.

FIG. 7 is a graph showing flow analysis of NK cells (CD3-CD16+ CD56+) after 7 days of induction culture of human peripheral blood mononuclear cells.

FIG. 8 is a graph showing the flow analysis of NK cells (CD3-CD16+ CD56+) after 14 days of induction culture of human peripheral blood mononuclear cells.

FIG. 9 is a graph showing flow analysis of NK cells (CD3-CD16+ CD56+) after 18 days of induction culture of human peripheral blood mononuclear cells.

FIG. 10 is the IFN-. gamma.content of collected NK cells in serum-free medium for 24h after induction culture for 14 days.

FIG. 11 is the cytotoxic effect of NK cells on K562 cells obtained after different time periods of induction culture of PBMC cells by the optimal culture protocol.

FIG. 12 is a graph showing the cytotoxic effect of collected NK cells on tumor cells after 14 days of induction culture.

Detailed Description

The invention is further described below with reference to the figures and examples.

The cytokines used herein, interleukin-2, interleukin-15 and 4-1BBL, were purchased from Beijing Erlu pharmaceutical Co., Ltd, the biological technologies Co., Ltd, monoclonal fluorescent antibodies used in flow cytometry detection, lymphocyte separation fluid, SCGM serum-free medium, CellGenix, Germany, and IFN-. gamma.ELISA detection kit, R & D biological technologies, were purchased from R & D Biotech, and non-radioactive cytotoxicity detection kit, for detecting the cytotoxic effect of NK cells on tumor cells, were purchased from Promega, USA.

The experiment was repeated with peripheral blood specimens from 6 normal volunteers, all achieving similar effects.

The first embodiment is as follows: induced expansion of NK cells

(1) Blood collection: collecting about 40ml of fresh anticoagulation blood;

(2) separation: centrifuging at 600g for 15 min, sucking the upper layer light yellow serum into a 50ml centrifuge tube, and separating PBMC by using a middle white membrane layer;

(3) inactivation of plasma: collecting serum, inactivating in 56 deg.C water bath for 30min, transferring blood plasma to a new 50ml centrifuge tube after the serum temperature is reduced to below 37 deg.C, storing in-20 deg.C refrigerator, and preparing NK cell culture solution;

(4) PBMC separation: transferring about 5ml of the white membrane layer obtained in the step (2) into 150 ml centrifuge tube, adding physiological saline to 20ml, and uniformly mixing; the cells were individually added to 4 15ml centrifuge tubes each containing 5ml of human lymphocyte cell lysate, taking care not to disrupt the liquid interface. Centrifuging at 400g for 20min (the acceleration and deceleration of the centrifuge are recommended to be adjusted to the lowest). The middle leucocyte layer is sucked and transferred into 150 ml centrifuge tube, washed twice with normal saline and counted, and each milliliter of anticoagulated peripheral blood should be about 1-2X 10⁶PBMC;

(5) incubation of the culture plate: adding 2ml PBS containing stimulating factors interleukin-15 (2 μ g/ml) and 4-1BBL (2 μ g/ml) into 6-well culture plate one day in advance, incubating at 4 deg.C, and washing off unfixed interleukin-15 and 4-1BBL with PBS;

(6) take about 4X 10⁷Adding 19ml of SCGM serum-free medium, 1ml of inactivated serum and 1 × 10 to PBMC⁴IL-2 of U, mixing, adding into incubatorThe cultured plates, 5ml per well, were cultured in an incubator (37 ℃ C., 5% CO)₂Concentration);

(7) supplementing culture solution to 3 days, adding 3ml of 1% serum-containing SCGM culture medium into each well, and continuing culturing;

(8) on day 4, the cells were harvested, centrifuged, the supernatant removed, suspended in 30ml of 1% serum SCGM medium, transferred to a T75 flask for further culture and IL-2, IL-15 and 4-1BBL were added to the flask to a final concentration of 500U/ml, the latter two being different concentration combinations as shown in Table 1.

(9) From day 5, half the fluid infusion every other day: SCGM medium supplemented with 1-2% serum, and IL-2(500U/ml), and IL-15 and 4-1BBL at different concentrations in combination (the combination of the concentrations is shown in Table one), at 37 deg.C, 5% CO₂The culture was continued in the cell culture chamber until day 18.

TABLE 1 (Unit: ng/ml)

(10) The cells cultured to day 18 of each group were collected and counted, and the results are shown in FIG. 1. As can be seen, after 18 days of culture of peripheral blood PBMC according to different protocols, more than 5X 10 cells were obtained starting from group 3⁹The number of cells obtained by the culture of groups 4 and 5 was not significantly increased compared to that of the cells obtained by the culture of groups 4 and 5, indicating that the number of cells obtained was not significantly increased as the concentration of IL-15 and 4-1BBL was increased after reaching 10 ng/ml. Therefore, group 3 was the most suitable culture protocol, i.e., the optimal concentrations for culturing cells with IL-15 and 4-1BBL were 10 ng/ml.

Example two: flow cytometry for detecting quantity content of NK cells

(1) According to the determination of the optimal culture protocol (the optimal concentration of IL-15 and 4-1BBL in the case of culturing cells is 10ng/ml), the cells in the optimal culture protocol are selected as the observation targets. Observing the cell morphology under an inverted phase contrast microscope, wherein the result is shown in FIG. 2, which shows that the cell morphology is circular, the volume is small, the cell brightness is good, and no particle is seen in the cell on the 0 th day of culture; at the 18 th day of culture, the results are shown in FIG. 3, and the cell growth map shows that the cells grow into clusters, part of the cells grow adherent to the walls, the cell morphology is irregular, the cell volume is larger than that of the common lymphocytes, and the transparency is good.

(2) Calculation of the number of cultured cells: cell viability was measured by total cell count and Taiwan phenol blue staining of cells cultured on

days

0, 3, 7, 9, 12, 14, 16 and 18. See fig. 4 and 5 for results: the initial cell number of the cultured cells was about 40X 10⁶The cell proliferation rate is increased at 7 days of induction culture, and the total cell number is continuously increased along with the time, and the total cell number reaches 4.2 multiplied by 10 at 14 days⁹And (4) respectively. The viable cell rate did not increase continuously with time after 3 days of culture, reached a maximum of 97.6% at day 14 and then began to decrease, indicating that more and more cells began to die and that the survival rate was only 90.3% by day 18 of culture (see figure 5). Therefore, the NK cell viability was the best when cultured up to day 14.

(3) The cells were collected on

days

0, 7, 14 and 18, respectively, and the collected cells were collected 10 per tube⁶Washing twice with 3ml PBS containing 2% fetal bovine serum, centrifuging for 300g × 5 min before each pouring, and draining the liquid in the tube mouth with filter paper after pouring; resuspending the cells with 100. mu.l PBS containing 2% fetal calf serum, adding 5. mu.l each of anti-human CD3 monoclonal antibody and anti-human CD56CD16 monoclonal antibody, mixing well, incubating for 30min at 4 ℃ in the dark; washing with 3ml PBS containing 2% fetal calf serum, centrifuging at 4 deg.C for 300g × 5 min, pouring, draining the liquid in the tube with filter paper, adding 400 μ l PBS containing 2% fetal calf serum to resuspend the cells, and detecting on a flow cytometer.

(4) Flow cytometry detection: at least 10000 cells are obtained from each tube by applying FACSDiva software, and the percentage content of NK cells in the total cells is calculated by taking CD3 negative and CD56CD16 double positive as the phenotypic characteristics of the NK cells. Results see FIGS. 6, 7, 8 and 9, where CD3FITC-A refers to anti-CD3FITC, CD16CD56PE-A refers to anti-CD16CD56 PE; the cell population in the upper left corner of the box in the figure represents NK cells, the percentage indicating their content in all cultured cells. In FIG. 6, it was found that NK cells were only about 10% in mononuclear cells before induction culture; with increasing induction time, theNK cell content in monocytes was 20% at day 7 (fig. 7), approximately 75% by day 14 (fig. 8), and approximately 80% by day 18 (fig. 9). In view of 40 × 10⁶NK content in PBMC was only about 10% (see FIG. 6), and the total number of cultured cells was 5.5X 10 by day 18⁹(see FIG. 4), the viable cell rate was about 90%, and the purity of NK cells therein was about 80% (see FIG. 8), indicating that NK cells had proliferated by thousands times. The above results indicate that the production and proliferation of NK cells can be effectively induced by culturing peripheral blood PBMC after adding interleukin-2, interleukin-15 and 4-1BBL to SCGM medium containing 1-2% human serum.

Example three: ELISA (enzyme-Linked immunosorbent assay) for detecting IFN-gamma content generated by NK (Natural killer) cells

1) Reagent:

(1) coating buffer (ph 9.60.05m carbonate buffer): NaCO₃1.59 g of NaHCO₃2.93 g, and distilled water was added to 1000 ml.

(2) Washing buffer (ph7.4 pbs): 0.15M KH2PO40.2 g, Na₂HPO₄·12H₂O2.9 g, NaCl8.0 g, KCl 0.2 g, Tween-200.05% 0.5ml, adding distilled water to 1000ml

(3) Diluting with Bovine Serum Albumin (BSA)0.1 g, and adding washing buffer solution to 100 ml; or 5-10% of serum such as sheep serum, rabbit serum and the like is mixed with the washing liquid for use.

(4) Stop solution (2M H)₂SO₄): 178.3ml of distilled water, 21.7ml of concentrated sulfuric acid (98%) were added dropwise.

(5) Substrate buffer (ph5.0 sodium dihydrogen phosphate-citric acid): 0.2M Na₂HPO₄(28.4 g/L) 25.7 ml; 24.3ml of 0.1M citric acid (19.2 g/L); 50ml of distilled water was added.

(6) TMB (tetramethylbenzidine) use solution: TMB (10mg/5ml absolute ethanol) 0.5ml substrate buffer (pH5.5)10ml, 0.75% H₂O₂32μl。

(7) ABTS use solution: ABTS 0.5mg, substrate buffer (pH5.5)1ml, 3% H₂O₂2μl。

(8) Antigens, antibodies and enzyme-labeled antibodies.

(9) And (5) detecting the sample.

2) Procedure for the preparation of the

(1) NK cells cultured to day 14 and peripheral blood PBMC were collected at 3X 10 each⁶The cells were resuspended and mixed well with 3ml of serum-free SCGM medium and added to 24-well plates. NK cells and PBMC each 3 wells, each 1 ml. Additional 3-well serum-free SCGM medium was added as a negative control. Culturing in incubator (37 deg.C, 5% CO)₂Concentration).

(2) After 24 hours of incubation, the culture supernatant (i.e., the sample to be tested) was collected and prepared for ELISA detection of various reagents.

(3) Coating: the antibody is diluted with 0.05M pH9.6 coating buffer to a protein content of 1-10. mu.g/ml. Mu.l of the solution was added to each reaction well of a 96-well polystyrene plate overnight at 4 ℃. The next day, the well was discarded, and the wells were washed 3 times with wash buffer (abbreviated as washing, the same below) for 3 minutes each.

(4) Sample adding: adding 100 μ l of diluted sample to be tested into the coated reaction well, and incubating at 37 deg.C for 1 hr. And then washed. Blank wells, negative control wells and standard curve wells were also prepared.

(5) Adding an enzyme-labeled antibody: to each reaction well, 0.1ml of a freshly diluted enzyme-labeled antibody (dilution after titration) was added. Incubating for 0.5-1 hour at 37 ℃, and washing.

(6) Adding a substrate solution for color development: and adding 100 mu l of TMB substrate solution prepared temporarily into each reaction hole, and keeping the temperature at 37 ℃ for 10-30 minutes.

(7) And (3) terminating the reaction: 50. mu.l of 2M sulfuric acid was added to each reaction well.

(8) Within 30min, the reaction plate is placed in an ELISA detector and programmed to detect at 450nm (410 nm if developed with ABTS), and the detector automatically generates a standard curve according to the standard curve hole and calculates the sample concentration. (ii) a

(9) The data were compiled to obtain IFN-. gamma.production by NK cells for 24h, and the results are shown in FIG. 9. As can be seen from the figure, the medium contained no IFN-. gamma.and thus the detected IFN-. gamma.content was secreted from PBMC or NK cells. 1X 10⁶The PBMC generate IFN-gamma of 74pg/ml after 24 h; 1X 10⁶Individual NK cells produced 3009pg/ml IFN- γ after 24h, significantly higher than the former. It says thatThe immune function of NK cells after induction culture is obviously improved.

Example four: determination of the most cytotoxic NK cells

(1) The effector cells in this assay were optimally cultured induced NK cells at different times. The target cells in the assay were K562 tumor cells.

(2) And (3) detecting the NK cell mediated cytotoxicity, wherein the detection plates are arranged as follows, and each group is provided with 3 multiple holes:

A. effector cell spontaneous LDH release: each concentration of effector cells to be used in the experimental wells was added to the wells containing the culture medium in 3 replicate wells to obtain effector cell spontaneous release values. The final volume must be the same as the experimental well (make up the volume with medium without cells);

B. experiment hole: 5000 target cells were added to all experimental wells of the U-bottom 96 well plate. Effector cells were added to the wells in 3 replicate wells and tested by adding effector cells cultured for various periods of time at an effective target cell ratio of 10: 1. The final volume of the target cells together was 100. mu.l/well;

C. target cell spontaneous LDH release: target cells were added to 3 replicate wells containing medium, and the final volume had to be equal to the experimental wells containing target and effector cells (volume adjusted with medium);

D. maximum LDH release from target cells: target cells were added to 3 replicate wells containing medium and the final volume had to be equal to the experimental wells. Mu.l of cell lysate (10X) was added per 100. mu.l of medium. Incubating the target cells in cell lysate for 45 minutes before harvesting the supernatant;

E. volume correction control: mu.l of cell lysate (10X) was added to 3 replicate wells containing 100. mu.l of medium (without cells). This control was used to correct for volume changes caused by the addition of cell lysate (10 ×);

F. background of the culture medium: add 100. mu.l of medium to 3 replicate wells. This control was used to correct for background absorbance caused by LDH activity in phenol red and serum-containing media;

the plates were centrifuged at G.1500rpm for 4 minutes to ensure adequate contact between effector and target cells.

(3) Cell culture and harvest of supernatant:

A. at 37 ℃ 5% CO₂The cell culture incubator of (1) incubates the cytotoxicity detection plate for 6 hours. To ensure sufficient time contact between target cells and effector cells;

B. 45 minutes before collecting the supernatant, 10. mu.l of cell lysate (10X)/100. mu.l of target cells were added to the maximum release pore of the target cells;

C. note that: if the target cells were not lysed completely (as determined by under-the-lens observation), 5. mu.l of cell lysate (10X) was added;

D. after 45 minutes of incubation, the plates were centrifuged at 1500rpm for 4 minutes;

E. measurement of LDH:

i. transfer 50. mu.l of supernatant from each well with a line gun to a new 96-well flat-bottom enzyme assay plate;

add 50. mu.l of the prepared substrate to each well of a 96-well enzyme assay plate containing the transferred sample supernatant. Incubating for 30 minutes at room temperature in the dark;

adding 50 μ l of stop solution to each well;

puncturing the large bubbles with a syringe needle, and measuring the absorbance at 490 or 492nm within 1 hour after the addition of the stop solution;

(4) calculation of experimental results:

A. subtracting the average value of the background light absorption values of the culture medium from the light absorption values of all the experimental wells, the target cell spontaneous LDH release holes and the effector cell spontaneous LDH release holes;

B. subtracting the mean value of the volume correction control light absorption value from the light absorption value of the maximum LDH release control of the target cells;

C. the percentage of cytotoxicity generated for each effect-to-target ratio was calculated by substituting the corrected values obtained in steps a and B into the following formula:

% cytotoxicity ═ 100 (experiment-effector cells spontaneous-target cells spontaneous) ÷ (target cells maximal-target cells spontaneous);

(5) from the calculation results, the cytotoxic effect of NK cells on K562 tumor cells at different culture times can be obtained when the effective target cell ratio is 10:1, and the results are shown in fig. 11: the NK cells can remarkably kill tumor cells when cultured by combining 500U/ml interleukin-2, 10ng/ml interleukin-15 and 10ng/ml 4-1BBL for 12 days, 14 days, 16 days and 18 days, and the cell killing rates are 55.7%, 60.3%, 55.5% and 49.4% respectively. These results indicate that the NK cells induced by the combination of IL-2, IL-15 and 4-1BBL with SCGM medium had the highest effect of killing tumor cells by 14 days, which is the optimum culture time.

Example five: detection of cytotoxic Effect of NK cells on various tumor cells

(1) The effector cells in this assay were the most optimal protocol for inducing NK cells at 14 days in culture. The target cells in the experiment were PANC-1 cells, U251 cells and K562 cells, all tumor cell lines.

(2) NK cell-mediated cytotoxicity was measured in the same manner as in example four. Several sets of effector cell ratios (1:1, 3:1, 10:1 and 30:1) were tested with different numbers of effector cells added.

(3) According to the calculation results, the cytotoxic effect of NK cells on tumor cells can be obtained, and the results are shown in FIG. 12: NK cells after combined culture for 14 days by using 500U/ml interleukin-2, 10ng/ml interleukin-15 and 10ng/ml 4-1BBL can obviously kill tumor cells, and the death rates of PANC-1 cells, U251 cells and K562 cells reach maximum values at a ratio of 30:1, and are respectively 50%, 60% and 84%. These results suggest that NK cells induced by the combination of interleukin-2, interleukin-15 and 4-1BBL with SCGM medium culture can effectively kill tumor cells, and that it can be considered for immunotherapy of tumor patients.

Claims

1. An in vitro induction amplification culture method of peripheral blood NK cells is characterized by comprising the following steps:

C. resuspending the mononuclear cells by using 38ml of SCGM culture medium, adding 2ml of inactivated autologous serum and 2 ten thousand units of recombinant human IL-2, uniformly mixing, adding into a culture plate, and placing into an incubator at 37 ℃ for standing culture; interleukin-15 and 4-1BBL required for culturing and activating NK cells are fixed in the culture plate;

D. on day 3, 20ml of SCGM medium containing 1-2% of autologous serum, 1 ten thousand units of IL-2, 10ng/ml of interleukin-15 and 10ng/ml of 4-1BBL were supplemented, and the culture was continued after mixing;

E. on day 4, the culture medium in the plate was transferred to a culture bag and further cultured, and 40ml of SCGM medium containing 1-2% of autologous serum and 2 ten thousand units of IL-2, 10ng/ml of interleukin-15 and 10ng/ml of 4-1BBL were supplemented to the culture bag every 3-4 days for a total of 18 days to obtain a total number of NK cells of 5.5X 10⁹And a viable cell rate of 90%, and wherein the purity of NK cells is 80%.

2. The method for culturing peripheral blood NK cells by in vitro induction and expansion according to claim 1, wherein: in the step B, the mononuclear cells collected from each 40ml of the peripheral blood sample are resuspended in 5ml of erythrocyte lysate and bathed in water at 37 ℃ for 1 minute, and the residual erythrocytes are completely lysed.

3. The method for culturing peripheral blood NK cells by in vitro induction and expansion according to claim 1, wherein: and in the step C, PBS containing interleukin-15 and 4-1BBL is added to the culture plate one day ahead for incubation and preservation at 4 ℃, and the unfixed interleukin-15 and 4-1BBL are washed away when the culture plate is used.

4. The method for culturing peripheral blood NK cells by in vitro induction and expansion according to claim 3, wherein: PBS containing 2. mu.g/ml interleukin-15 and 2. mu.g/ml 4-1BBL was added to the plate one day in advance and incubated at 4 ℃ for storage.